[unreadable] [unreadable] MicroRNAs (miRNAs) are small, non-coding RNAs that are indispensable regulators of gene expression. Studies of model organisms have determined that worms, fish, and mice are not able to live without miRNAs. However, little is known about the specific biological pathways and processes regulated by the vast majority of individual miRNAs during animal development. To this end, we have isolated mutant alleles in nearly all miRNA genes in Caenorhabditis elegans. Strikingly, the majority of individual miRNAs are not essential for development or viability. The goal of research in this proposal is to identify a network of genetic interactions between individual miRNAs and other miRNAs, signaling components, and transcription factors. We will focus on those miRNAs that show sequence conservation between worms and mammals. We will use a genetic approach to identify regulatory pathways controlled by miRNAs during C. elegans development. (Aim 1) The hypothesis will be tested that distinct miRNAs function together to regulate cell differentiation, proliferation or survival, possibly by repressing a shared set of target mRNAs. We will analyze the phenotypes of worms with mutations in individual miRNA genes in a sensitized genetic background in which the activity of all miRNAs is reduced. (Aim 2) We will test the hypothesis that miRNAs function to modulate or reinforce signaling pathways, possibly functioning to establish a cell's sensitivity or response to signaling molecules in order to confer robustness of developmental signaling pathways. We will use RNAi to systematically test individual miRNA mutants for genetic interactions with all genes identified as signaling components in the C. elegans genome. (Aim 3) We will test the hypothesis that miRNA-mediated post-transcriptional regulation functions in concert with transcriptional control of shared downstream targets. We will use RNAi to test for functional genetic interactions between individual miRNA genes and all transcription factors in C. elegans. This research will advance our understanding of the principles underlying miRNA action in a cell and will identify functional connections between miRNAs, signaling pathways and transcription factors. Determination of the normal pathways regulated by miRNAs is a critical initial step towards identifying causal relationships between miRNA expression patterns and cancer initiation or progression. [unreadable] [unreadable] Public Health Relevance: MicroRNAs (miRNAs) are small regulatory RNAs that have been implicated in the development or progression of multiple types of human cancers including lung, colorectal, and breast cancer. However, it is not yet clear whether changes in miRNA expression are a cause or consequence of carcinogenesis. In order to elucidate a potential causative role of miRNAs in carcinogenesis, we will use genetic analysis of miRNA mutant C. elegans in order to identify the normal function of miRNAs in cellular processes. [unreadable] [unreadable] [unreadable]